The overall goal of this proposal is to understand how papillomviruses replicate their genomes. Papillomaviruses have in recent years emerged as a very important causal agent in human disease. These viruses as part of their normal life cycle infect and transform cells in the epithelium causing benign tumors that with a low, but significant, frequency can become malignant. A deeper understanding of the life cycle in general, and DNA replication in particular, is of critical importance for the understanding of the disease, its transmission and ultimately for the development of effective therapeutic measures. We are studying DNA replication of papillomaviruses in vivo and in vitro. We are combining genetic biochemical and structural analyses of the viral proteins and sequence elements that are required for viral DNA replication. In this proposal we will continue our in depth analysis of the papillomavirus replicon with emphasis on the assembly pathways responsible for the ordered recognition, distortion and unwinding of the viral origin of replication. We propose to (i) characterize the transition from the E12E22 complex to the E1 DT. (ii) To characterize the E1 DT and to analyze its transition to the DH. (iii) To investigate the properties of the E1 DH and to characterize its activity. (iv) To analyze nuclear extract activated E1 DH formation. PUBLIC HEALTH RELEVANCE: Papillomaviruses are very important causative agents of human disease. The viral DNA replication machinery presents one of the few potential targets for drug therapy. Our studies, which are directed towards understanding the viral DNA replication machinery in detail will provide new such potential targets.